Compositions and methods for controlling stem cell and tumor cell differentiation, growth, and formation

ABSTRACT

The present invention relates to the use of self-assembling peptide amphiphiles to prevent tumor formation by transplanted stem cells. The present invention further relates to the use of self-assembling peptide amphiphiles to treat cancers.

This application claims priority to provisional patent application Ser.No. 60/532,249, filed Dec. 23, 2003, which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the use of self-assembling peptideamphiphiles to prevent tumor formation by transplanted stem cells. Thepresent invention further relates to the use of self-assembling peptideamphiphiles to treat cancers.

BACKGROUND OF THE INVENTION

Embryonic stem cells are pluripotent and can give rise to virtually allcell lineages in the body. Thus there is a great clinical potential forusing embryonic stem cells to replace damaged cells in any given organor tissue. However, embryonic stem cells can be tumorigenic. Therefore,a major concern for using embryonic stem cells is tumor formation aftergrafting. What is needed are compositions and methods useful inpreventing tumor formation after embryonic stem cell grafting.

SUMMARY OF THE INVENTION

The present invention relates to the use of self-assembling peptideamphiphiles to prevent tumor formation by transplanted stem cells. Thepresent invention further relates to the use of self-assembling peptideamphiphiles to treat cancers.

For example, the present invention provides a method, comprising thesteps of a) providing a stem cell, a peptide-amphiphile composition, anda subject; and b) administering the stem cell and the peptide-amphiphilecomposition to the subject (e.g., under conditions such that tumors arenot formed or tumor growth or formation is reduced as compared to asubject undergoing the same treatment in the absence of thepeptide-amphiphile composition). In some preferred embodiments, the stemcell comprises an embryonic stem cell or an adult stem cell. In someembodiments, the peptide-amphiphile composition comprises a hydrophobiccomponent, a peptide or peptide-like component, and a bioactive epitopesequence. In some embodiments, the bioactive epitope sequence is IKVAV.In some embodiments, the peptide-amphiphile composition is a gel. Inpreferred embodiments, the peptide-amphiphile composition and the stemcells are mixed prior to the administering step. While the presentinvention is not limited by the nature of the subject, in someembodiments, the subject is suffering from ischemia.

The present invention also provides methods comprising the steps of: a)providing a peptide-amphiphile composition; and a subject, wherein thesubject has a tumor; and b) contacting the peptide-amphiphilecomposition with the subject under conditions such that the subject'stumor is decreased in size or such that additional tumor formation ormetastasis is reduced or prevented.

The present invention further provides compositions comprising one ormore stem cells and a peptide-amphiphile composition. The compositionsmay comprise kits for research or therapeutic applications. In someembodiments, the stem cells and peptide-amphiphile composition are incontact with one another. In other embodiments, the stem cells andpeptide-amphiphile composition are separate (e.g., are in separatecontainers in a kit).

DESCRIPTION OF THE FIGURE

FIG. 1 shows the protection of mice from tumor formation after embryonicstem cell transplantation.

Definitions

As used herein, the term “mesodermal cell line” means a cell linedisplaying phenotypic characteristics associated with mesodermal cells.

As used herein, the term “endodermal cell line” means a cell linedisplaying phenotypic characteristics normally associated withendodermal cells.

As used herein, the term “ectodermal cell line” means a cell linedisplaying phenotypic characteristics normally associated withectodermal cells.

As used herein, the term “pluripotent” means the ability of a cell todifferentiate into multiple different types of cells (e.g., terminallydifferentiated cells). For example, pluripotent cells include those thatcan differentiate into the three main germ layers: endoderm, ectoderm,and mesoderm.

As used herein, the terms “transplant cells” and “graft material” referbroadly to the component (e.g., tissue or cells) being grafted,implanted or transplanted. As used herein, the term “transplantation”refers to the transfer or grafting of tissues or cells from one part ofa subject to another part of the same subject, or to another subject, orthe introduction of biocompatible materials into or onto the body. Asused herein, in some embodiments, a transplanted tissue may comprise acollection of cells of identical or similar composition, or derived froman organism (e.g., a donor), or from an in vitro culture (e.g., a tissueculture system).

The term “recipient of transplanted cells” as used herein, refersbroadly to a subject undergoing transplantation and receivingtransplanted cells.

As used herein, the term “cell culture” refers to any in vitro cultureof cells, including but not limited to continuous cell lines (e.g., withan immortal phenotype), primary cell cultures, and finite cell lines(e.g., non-transformed cells).

The term “in vitro” refers to an artificial environment and to processesor reactions that occur within an artificial environment. The term “invivo” refers to the natural environment (e.g., an animal or a cell) andto processes or reactions that occur within a natural environment. Thedefinition of an in vitro versus in vivo system is particular for thesystem under study. As used herein, an in vitro system refers to studiesof cells or processes in an artificial environment, such as in tissueculture vessels and apparatus, whereas study of the same system in an invivo context refers to the study of cells or processes within anorganism, such as a rat or human.

As used herein, the term “primary cell” or “primary culture” refers to acell or a culture of cells that have been explanted directly from anorganism, organ, or tissue. Primary cultures are typically neithertransformed nor immortal.

The term “tissue culture” as used herein, refers to a collection oftechniques for the growth and maintenance of cells in the laboratory.Such techniques may involve tissue culture dishes or other vessels,incubators and sterility containment devices, as known in the art.

As used herein, the term “exogenous” is used interchangeably with theterm “heterologous” refer to a substance coming from some source otherthan its native source. For example, the terms “exogenous protein,” or“exogenous cell” refer to a protein or cell from a non-native source orlocation, and that have been artificially supplied to a biologicalsystem. In contrast, the terms “endogenous protein,” or “endogenouscell” refer to a protein or cell that are native to the biologicalsystem, species or individual.

As used herein, the term “stem cells” refers to cells that canself-renew and differentiate into multiple lineages. Stem cells may bederived, for example, from embryonic sources (“embryonic stem cells”) orderived from adult sources. For example, U.S. Pat. No. 5,843,780 toThompson describes the production of stem cell lines from human embryos.PCT publications WO 00/52145 and WO 01/00650 describe the use of cellsfrom adult humans in a nuclear transfer procedure to produce stem celllines.

Examples of adult stem cells include, but are not limited to,hematopoietic stem cells, neural stem cells, mesenchymal stem cells, andbone marrow stromal cells. These stem cells have demonstrated theability to differentiate into a variety of cell types includingadipocytes, chondrocytes, osteocytes, myocytes, bone marrow stromalcells, and thymic stroma (mesenchymal stem cells); hepatocytes, vascularcells, and muscle cells (hematopoietic stem cells); myocytes,hepatocytes, and glial cells (bone marrow stromal cells) and, indeed,cells from all three germ layers (adult neural stem cells).

The terms “embryonic stem cell” (“ES cell”) refer to cells derived frommammalian blastocysts, which are self-renewing and have the ability toyield many or all of the cell types present in a mature animal. Humanembryonic stem cell lines suitable for use with the methods andcompositions of the present invention include but are not limited tothose produced by the following institutions: BresaGen, Inc., Athens,Ga.; CyThera, Inc., San Diego, Calif.; ES Cell International, Melbourne,Australia; Geron Corporation, Menlo Park, Calif.; Göteborg University,Göteborg, Sweden; Karolinska Institute, Stockholm, Sweden; Maria BiotechCo. Ltd.—Maria Infertility Hospital Medical Institute, Seoul, Korea;MizMedi Hospital—Seoul National University, Seoul, Korea; NationalCentre for Biological Sciences/Tata Institute of Fundamental Research,Bangalore, India; Pochon CHA University, Seoul, Korea; Reliance LifeSciences, Mumbai, India; Technion University, Haifa, Israel; Universityof California, San Francisco, Calif.; and Wisconsin Alumni ResearchFoundation, Madison, Wis. The human ES cells listed on the HumanEmbryonic Stem Cell Registry to be created by the National Institutes ofHealth find use in the methods and compositions of the presentinvention. However, human ES cells not listed on the NIH registry arealso contemplated to find use in embodiments of the present invention(e.g., when it is desirable to prevent ES contamination withnonhuman-derived materials).

As used herein the term “feeder cells” refers to cells used as a growthsupport in a tissue culture system. In preferred embodiments, the term“feeder cells” refers to embryonic “striatum cells,” while in otherembodiments the term “feeder cells” refers to stromal cells.

DETAILED DESCRIPTION OF THE INVENTION

Pluripotent stem cells such as embryonic stem cells (ESCs) candifferentiate into a variety of cell types (e.g., all of the cell types)of the body and may potentially be used to repair damaged organs ortissues. However, the clinical utility of embryonic stem cells atpresent is very limited because they develop teratomas aftertransplantation into recipients. The methods of the present inventionprovide a novel approach involving association (e.g., co-injection) ofembryonic stem cells with a self-assembling gel to prevent tumorformation by embryonic stem cells or other stem cells. Experimentsconducted during the course of development of the present inventiondemonstrated protection against tumor formation after transplantationwith the use of the gel. The present invention thus provides methods foruse (e.g., research use, drug screening, therapeutic use) of embryonicstem cells for many types of disorders of different organs.

The methods of the present invention are suitable for use with a varietyof stem cells including, but not limited to, embryonic stem cells andadult stem cells. Embryonic stem cells may be obtained from a variety ofsources including, but not limited to, embryonic stem cell lines andembryonic germ cell lines derived from primordial germ cells (PGCS)cells isolated, according to one embodiment, from gonadal tissues,genital ridges, mesenteries or embryonic yolk sacs of human embryos (seee.g., U.S. Pat. No. 6,562,619). Embryonic stem cells may also beobtained from commercial or research sources. Adult stem cells may bederived from a variety of cell types, including, but not limited to,those disclosed above.

In some preferred embodiments, the present invention utilizespeptide-amphiphile compositions to prevent tumor formation by embryonicstem cells. Exemplary peptide-amphiphile compositions are described inWO 03/070749 and WO 03/084980; herein incorporated by reference in theirentireties. In some embodiments, the composition is a gel.

The peptide-amphiphile (PA) compositions used in the present inventioncan be synthesized using preparatory techniques well-known to thoseskilled in the art—preferably, by standard solid phase chemistry, withalkylation or other modification of the N-terminus of the peptidecomponent with a hydrophobic moiety, mono or di-alkyl moieties attachedto the N- or C-termini of peptides may influence their aggregation andsecondary structure in water in both synthetic and natural systems. Ahydrophobic, hydrocarbon and/or alkyl tail component with a sufficientnumber of carbon atoms coupled to an ionic peptide having a preferencefor beta-strand conformations can in certain embodiments be used tocreate an amphiphile that assembles in water into nanofiber structures.The amphiphile's overall conical shape can also have an effect on suchassemblies. Self-assembling can also be triggered by body fluid (e.g.,cerebral spinal fluid).

In some embodiments, the peptide amphiphile compound/composition has 1)a hydrophobic component and 2) a peptide or peptide-like componentfurther including a bioactive epitope sequence. In various preferredembodiments, the hydrophobic component of such a compound or compositionis of sufficient length to provide amphiphilic behavior and nanofiberassembly/formation in water or another polar solvent system. Typically,such a component may be about a C6 or greater hydrocarbon moiety,although other hydrophobic, hydrocarbon and/or alkyl components could beused as would be well-known to those skilled in the art to providesimilar structural or functional effect. Such hydrophobic componentsinclude, without limitation, cholesterol, biphenyl and p-aminobenzoicacid.

In some embodiments, the bioactive epitope is an IKVAV sequence. IKVAVis a laminin sequence known to interact with mammalian neurons. IKVAVpromotes neurite outgrowth in mammalian neurons. The present inventionis not limited to the use of IKVAV. Other suitable bioactive epitopesfind use in the methods of the present invention.

Peptide components of this invention preferably comprisenaturally-occurring amino acids. However, incorporation of knownartificial amino acids such as beta or gamma amino acids and thosecontaining non-natural side chains, and/or other similar monomers suchas hydroxyacids are also contemplated, with the effect that thecorresponding component is peptide-like in this respect.

In some embodiments, the PA compositions form a sol-gel systemincluding 1) a polar or aqueous solution and/or containing of one ormore of the amphiphile compounds or compositions described herein, and2) a factor or reagent sufficient to induce assembly, agglomeration ofgelation under neutral or physiological conditions. Such gelation and/orself-assembly of various PA compositions into micellular nanofibers canbe achieved under substantially neutral and/or physiological pHconditions through drying, introduction of a mono- or multivalent metalion and/or the combination of differently charged amphiphiles.

Experiments conducted during the course of development of the presentinvention demonstrated that transplantation of embryonic stem cellsimproves functional outcome after brain ischemia in rats. However, about20˜30% of the recipients died of brain tumors. In contrast, when thepeptide amphiphiles was co-injected with the embryonic stem cells, theanimals did not develop tumors. Accordingly, in some embodiments, thepresent invention provides methods of preventing tumor formation uponintroduction of stem cells (e.g., embryonic stem cells or ESCs) into asubject. In some embodiments, the gels are co-injected during ESCtransplantation in any organ to prevent teratoma formation. In someembodiments, the gels and the stem cells are mixed prior to injection.Any suitable stem cell or peptide-amphiphile composition may beutilized. Preferred compositions are non-toxic. A variety of stem cellsmay be utilized, depending on the application.

In other embodiments, the peptide-amphiphile compositions are used forcancer treatment in situ. For example, in some embodiments, peptideamphiphile (e.g., those described herein) are injected directly at thesite of a tumor or cancer. The treatment may be repeated as needed untilthe tumor is reduced in size or eliminated.

EXPERIMENTAL

The following examples are provided in order to demonstrate and furtherillustrate certain preferred embodiments and aspects of the presentinvention and are not to be construed as limiting the scope thereof.

Example 1

Protection of ESC-Transplanted Mice Against Tumor Formation.

This Example describes the protection of embryonic stem cell (ESC)transplanted mice against tumor formation in the presence of peptideamphiphiles (IKVAV-peptide amphiphile).

A. Methods

Animal Model of Focal Brain Ischemia

Under anesthesia (Avertin, 0.2 ml/10 gram body weight, i.p.), anincision was made between left ear and eye in adult female mice (129 sv,4-8 w). A craniotomy was performed, and the middle cerebral artery wasexposed. Thereafter, the left middle cerebral artery was cauterized, andthe incision was sutured by a 3-0 silk surgery suture.

Embryonic Stem Cell Preparation

Embryonic stem cells (R1, a cell line derived from 129sv mice) werepropagated in DMEM defined medium (20% fetal bovine serum, 0.1 mM ofnon-essential amino acids, 50 μg/ml of penicillin-streptomycin, 0.1 mMof L-glutamine, 1000 U/ml of leukemia inhibitory factor, 100 μM ofβ-mercaptoethanol). The cells were harvested with 0.05% trypsin-EDTA.After washing with HBSS (Hanks' Balanced Salt Solution) twice, the cellswere then re-suspended in IKVAV-PA or glucose (vehicle for IKVAV-PA).The cell concentration was prepared as 50˜100,000 cells/μl.

Transplantation

One week after brain ischemia, the mice were anesthetized with Avertin.The animals were fixed in a KOPF brain frame, and 2 μl of embryonic stemcells were stereotoxically injected into the left cortex surrounding theinfarction by using a 10-μl Hamilton microsyringe. Two sites wereselected for grafting: 1) 0.26 mm rostal to the bregma, 2.5 mm lateralto the midline, 1.5 mm ventral to the dura; 2) 2.06 mm caudal to thebregma, 2.5 mm lateral to the midline, 1.5 mm ventral to the dura. Thetooth bar was set at −2. The cells were injected through 2 minutes andthe cannula was kept in situ for 5 minutes before removal.

Tumor Formation

After grafting, the mice were monitored every week. Dead animals withlarge brain tumors were removed out from their cage.

B. Results

ESCs were suspended with the self-assembling IKVAV-peptide amphiphile(gel) or with vehicle before grafting. Female adult 129SV mice weresubjected to permanent middle cerebral artery occlusion to induce brainischemia. One week after brain ischemia, the mice were randomly dividedinto nine groups (5 mice/group). Two microliters of ESC suspension(50˜100,000 cells/μl, 2 sites/brain) were stereotaxically injected intothe cortex surrounding the infarctions. The results are shown in FIG. 1.R1 is the wild type strain of mouse for the embryonic stem cells used inthese experiments. #6, #41, #158 and #168 are clones of SOX1promoter-eGFP engineered R1 ESCs. RA (retinoic acid) has been reportedto induce embryonic stem cell differentiation into neural cells. Asearly as three weeks after ESC transplantation, the animals without thegel started dying from tumors. All of the mice that received #41 ESCswithout RA treatment died at 3 weeks after grafting. Three months aftertransplantation, 60%˜80% of ESC grafted mice died of brain tumor. Incontrast, the mice that grafted with ESC+IKVAV-PA all survived at 3months after grafting.

ESCs were also found to survive after seeding in vitro with IKVAV-PA.ESCs were seeded with IKVAV-AP (4×10⁴ cells/ml) and they survived well 2days after seeding. ESC viability was assayed 7 days after seeding withIKVAV-PA or seeding on PDL coated cover slips using fluorescencemicroscopy. These data indicated that IKVAV-PA was not cytotoxic toembryonic stem cells. In other words, IKVAV-PA did not kill theembryonic stem cells before and after transplantation. ESCs were shownusing fluorescence microscopy to differentiate into neuronal like cells3 weeks after seeding in IKVAV-PA.

Example 2

Transplantation of Glioma Cells (9L) with Self-Assembling Gel Under theSkin

This example describes the use of peptide-amphiphile gels to inhibittumor formation in vivo.

Preparation of Glioma Cells In Vitro

Gliosarcoma (9L) cells, derived from Fischer 344 rats, are grown in DMEMmedium with 10% fetal bovine serum, and are collected with 0.05%trypsin-EDTA. The cells are washed with HBSS twice and 10⁷ cells arere-suspended with 300 μl IKVAV-PA or glucose (vehicle control) or HBSS.

Transplantation of Glioma Cells with Self-Assembling Gel

Tumor cells are suspended with IKVAI-PA or glucose. The cells are theninjected subcutaneously in the shaved left flanks of anesthetized rat(female Fischer 344, 100˜150 g. Measurement of tumor formation isperformed weekly.

Transplantation of Glioma Cells Followed by Self-Assembling Treatment

Glioma cells are re-suspended with HBSS at a concentration of 10⁷cells/300 μl, and are then injected into the shaved right flanks ofFischer 344 rats under anesthesia. Three weeks after grafting, the ratsare randomly divided into two groups. One group of the grafted ratsreceives IKVAV-PA injection (5 sites: 4 in the periphery and one in thecentral of the tumor; 50 μl/site). In the control group, the same volumeof glucose is injected into the tumor. The peptide or glucose is giventwice a week. Tumor size is measured once a week.

1. A method, comprising: a) providing i) a stem cell; ii) apeptide-amphiphile composition; and iii) a subject; and b) administeringsaid stem cell and said peptide-amphiphile composition to said subject.2. The method of claim 1, wherein said stem cell is an embryonic stemcell.
 3. The method of claim 1, wherein said peptide-amphiphilecomposition comprises a hydrophobic component, a peptide or peptide-likecomponent, and a bioactive epitope sequence.
 4. The method of claim 3,wherein said bioactive epitope sequence is IKVAV.
 5. The method of claim1, wherein said peptide-amphiphile composition is a gel.
 6. The methodof claim 1, wherein said peptide-amphiphile composition and said stemcells are mixed prior to said administering.
 7. The method of claim 1,wherein said subject is suffering from ischemia.
 8. The method of claim1, wherein said stem cells are adult stem cells.
 9. A method,comprising: a) providing i) a peptide-amphiphile composition; and ii) asubject, wherein said subject has a tumor; and b) contacting saidpeptide-amphiphile composition with said subject under conditions suchthat said subject's tumor is decreased in size.
 10. The method of claim9, wherein said peptide-amphiphile composition comprises a hydrophobiccomponent, a peptide or peptide-like component, and a bioactive epitopesequence.
 11. The method of claim 10, wherein said bioactive epitopesequence is IKVAV.
 12. The method of claim 9, wherein saidpeptide-amphiphile composition is a gel.
 13. The method of claim 9,wherein said contacting said peptide-amphiphile composition with saidsubject comprises contacting said composition directly with the site ofsaid tumor.
 14. A composition comprising a stem cell and apeptide-amphiphile composition.